Expression of nebulette during early cardiac development

Abstract

Nebulette, a cardiac homologue of nebulin, colocalizes with alpha-actinin in the pre-myofibrils of spreading cardiomyocytes and has been hypothesized to play a critical role in the formation of the thin-filament-Z-line complex early during myofibrillogenesis. Data from mesodermal explants or whole tissue mounts of developing hearts suggest that the pattern of myofibrillogenesis in situ may differ from observations of spreading cardiomyocytes. To evaluate the role of nebulette in myofibrillogenesis, we have analyzed the expression of nebulette in chicken heart rudiments by immunoblots and immunofluorescence. We detect the 110 kDa nebulette in heart rudiments derived from stage 9-10 using the anti-nebulin mAb, N114, or polyclonal anti-nebulette Abs by immunoblotting. Immunofluorescence analysis of explants stained with anti-nebulette and anti-alpha-actinin Abs demonstrates that both proteins localize along actin filaments in punctate to continuous manner at early stages of cardiac development and later give rise to striations. In both cases, the punctate staining had a periodicity of approximately 1.0 microm indicating a pre-myofibrils distribution at the earliest time points examined. We demonstrate that nebulette is indeed associated with premyofibrils in very early stages of myofibrillogenesis and suggest that nebulette may play an important role in the formation of these structures.

Nebulette interacts with filamin C

“…Filamin C [Lu et al, 2003], N-RAP [Carroll and Horowits, 2000], and a-actinin [Rhee et al, 1994] are all found in structures referred to as premyofibrils in differentiating and spreading cardiomyocytes. We have previously demonstrated that nebulette is also a component of the premyofibrils in spreading [Moncman and Wang, 1995] and differentiating [Esham et al, 2007] cardiomyocytes. Although it is beyond the scope of this initial report, it is interesting to speculate that competitive interactions between nebulette and N-RAP may be important for sorting filamin C both to its costameric and intercalated disk localizations and to its mature myofibril localization.…”

Nebulette is a powerful cytolinker organizing desmin and actin in mouse hearts

“…On the other hand, the highest degree of homology (60–85%) of nebulette to nebulin is in its 23 central 35-residue nebulin-like single modules (Moncman and Wang, 1995). Besides playing important roles early in cardiac development, nebulette, like nebulin, binds and stabilizes actin filaments (Jin and Wang, 1991; Moncman and Wang, 1999; Ogut et al , 2003; Esham et al , 2007). Coincidentally, abnormally short actin filaments are prevalent in ex vivo cardiomyocytes overexpressing the nebulette serine-rich and SH3 domain or the mutant desmin E245D and in skeletal muscles from nebulin knockout mice (Li et al , 2004; Bang et al , 2006; Conover et al , 2009).…”

“…We investigated the possibility that the similar domain layout of nebulin and nebulette and their shared roles in mechanosensing and force generation and transmission may be related by a shared direct interaction with the IF cytoskeleton. Of note, both nebulette and desmin are expressed at high levels at early stages during cardiac development (Holtzer et al , 1982; Esham et al , 2007). We hypothesized that the cardiac-specific protein nebulette, besides interacting with several sarcomeric proteins, would bind desmin directly and, furthermore, that binding of nebulette would affect the assembly of desmin filaments in vitro.…”

Nonmuscle myosin IIB, a sarcomeric component in the extraocular muscles

“…In both cardiac and somite-derived skeletal muscles, nmMyH IIB is associated with the pre-myofibrils of differentiating muscle cells [39]. In addition to nmMyH IIB, the pre-myofibrils contain sarcomeric α-actinin [39] and in cardiac muscle, nebulette [20, 40]. With the addition of titin into the complex, the nonmuscle myosin is gradually replaced by sarcomeric myosin isoforms [39].…”